Effect of Seed Priming on Biochemical Changes in Fresh and ...
Transcript of Effect of Seed Priming on Biochemical Changes in Fresh and ...
Journal of Agricultural Studies
ISSN 2166-0379
2017, Vol. 5, No. 3
http://jas.macrothink.org 62
Effect of Seed Priming on Biochemical Changes in
Fresh and Aged Seeds of Cucumber
Pratima Pandey (Corresponding author)
Nepal Agricultural Research Council, Kathmandu.
E-mail: [email protected]
K Bhanuprakash & Umesha
Indian Institute of Vegetable Research, Bangalore.
Received: August 4, 2017 Accepted: August 18, 2017 Published: September 8, 2017
doi:10.5296/jas.v5i3.11637 URL: https://doi.org/10.5296/jas.v5i3.11637
Abstract
The present investigation was conducted at the laboratory of Seed Science and Technology,
Indian Institute of Horticultural Research, Hessaraghatta, and Bangalore, India during March
2014 to September 2014. The objective of the study was to identify effect of seed priming on
biochemical changes in cucumber seeds. This study showed that the enhancement in seed
viability and vigor in primed seeds was due to low membrane injury coupled with high
enzyme activities (dehydrogenase and amylase). The priming with KH2PO4 and K2HPO4
reduced the electrical conductivity (EC) significantly in seed. With seed priming with
KH2PO4 10-3
M there was 44 and 45% lower EC values compared to unprimed. This priming
with KH2PO4 10-3
M and K2HPO410-3
M reduced seed leachate values indicating priming
initiates the process of repair by stabilizing membrane integrity. priming significantly
increased dehydrogenase enzyme in low vigor seeds was noticed. These changes are to the
tune of 16 and 21 per cent due to priming with K2HPO410-3
M and KH2PO410-3
M,
respectively in cucumber. Similarly, higher amylase activity was noticed upon priming in low
vigor seeds These are to the extent of 2.1, 1.94 folds higher due to priming with
K2HPO410-3
M and KH2PO410-3
M, respectively, in cucumber and 1.4 & 1.4 folds higher due
to priming with K2HPO410-3
M .Alteration in protein profiles in low vigor seeds compared to
high vigor seeds was noticed in the crops. The disappearance of some low molecular weight
proteins in low vigor seeds might be the reason for lesser performance of these seeds. Seed
priming although not restored all the lost proteins, but induced synthesis of some other
proteins which probably might helped in restoration of germination ability and vigor. Esterase
Isozyme profiles also showed variation in their number and intensity in less vigor seeds.
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Primed seeds further exhibited tolerance to abiotic stress condition (high temperature and
water stress) as evident from higher germination compared to unprimed. Besides these
studies, some advanced studies were initiated in the present investigation to identify changes
in proteome profiles and isocitratelyase gene expression in high vigor and low vigor seeds
during germination.
Keywords: seed ageing, seed priming, biochemical, enzymes
1. Introduction
Cucumber (Cucumissativus L.) is one of the most important cucurbitaceous vegetables grown
throughout the world and ranks fourth after tomato, cabbage and onion. The crop has a wide
range of utilities such as pickling, salad, cooked vegetable as an ingredient in Unani
medicines and cosmetics. The world average productivity of cucumber is 16.53 t/ha but on
the contrary it is only 6.67 t/ha (Anon, 2004) in India. The wide gap in productivity can be
attributed to the lack of availability of quality seeds of high yielding varieties, promising
hybrids and parthenocarpic varieties suited for glass or poly house cultivation. Seed quality is
one of the key factors affecting the successful farming, but this seed trait inevitably declines
during prolonged storage. Cucumber is a thermophilic crop which requires relatively high
temperatures during its life cycle and generally seeds of cucurbits require relatively high
temperature for successful germination and seedling emergence (Hegarty, 1973). Optimum
seed germination and seedling growth in cucumber occur at 20-250C. Poor seed germination
is a common phenomenon at sub optimal temperatures which causes a great concern for
cucumber growers. According to McDonald (1999), primed seeds could shorten their steps of
DNA and RNA synthesis. Consequently, seeds physiology tends to be closer to germination
than the unprimed seeds. On the other hand, Higher rates of germination index (GI) might be
due to the result of recovered development of genetic repair mechanisms during priming
operations (Bradford,1986).Delayed and reduced germination and seedling emergence cause
non- uniformity in stand establishment and tender seedling development susceptible to soil
borne pathogens for long period. Cucumber plants are susceptible to chilling injury in fields
of prolonged low temperatures below 550F (12.8
0C) which results in poor seedlings of
splitting, water soaked lesions and decay. One of the major constraints in cucumber
cultivation is the poor stand establishment at sub optimal temperature (<200C). Several
priming treatments have been reported to enhance germination under stressful environmental
conditions like low temperature, moisture stress and saline soil beds and even to improve
poor quality seeds. The terms ‘seed priming’, ‘seed enhancement’ or ‘seed invigoration’
represents a series of treatments applied to a given seed lot in order to improve its
germination, uniform emergence of seedlings and yield. Seed priming is a pre-sowing
treatment that involves controlled hydration of seeds, sufficient to allow pre germinative
metabolic events to take place place and to restrict radical protrusion through the seed coat
(Heydecker et al., 1973). Seed priming enables a relatively prolonged Phase II, compared to
the water absorption of un- primed seeds in a typical germination process, which presumably
includes specific mechanism(s) that promotes rapid and uniform germination (Nascimento et
al., 2001) The This technique has been used in some vegetables seeds including cucumber to
augment the germination rate, total germination and seedling uniformity etc., mainly under
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unfavorable environmental conditions. It is a useful technique to exploit seed potential in arid
and desert ecosystem. Seed priming is a low cost technology in which controlled hydration of
seeds followed by re-drying is done to break dormancy, improve germination and stand
establishment (Afzal et al., 2009). Another benefit of seed priming is that damaged and poor
quality seeds float when they are soaked during priming, making it easier for farmers to
discard low quality seeds (Farooq and Basra, 2006). The knowledge gained on the repair
mechanisms that take place upon various priming treatments has been used in many crops of
seed industry. Hydro priming is nothing but soaking seeds in water for a precise time
followed by re-drying. Besides, benefits of seed priming are used on pre-enlargement of
embryo (Bradford et al., 1990) and extension speed of metabolites (Basra et al., 2005).
Kazem et al. (2008) evaluated the effects of hydro priming, halo- priming (solutions of 1.5 %
KNO3 and 15 mScm-1 NaCl) and osmopriming (PEG 6000 @ -0.8 MPa) on seedling vigour
and field establishment of lentil. Analysis of variance of laboratory data showed hydro
priming significantly improved the imbibition rate, germination rate, seedling vigour index,
shoot, root and seedling dry weights and reduced electrical conductivity of seed leachates,
compared to other seed treatments. Osmopriming involves soaking seeds in aerated water
potential of different osmotica (Polyethylene glycol, mannitol, sorbitol, glycerol, etc.,) to
control the amount of water imbibed by seeds. Chemo priming is soaking seeds in various
inorganic salt solutions like KCl, KNO3, CaCl2, KH2PO4, etc.. the results for germination
percentage confirmed the findings of Warley and Fernando (2004) who reported that priming
improved the germination percentage and germination rate of un-aged and aged seeds of
muskmelon.
2. Material and Methods
The present investigation on seed invigoration aspects of cucumber was conducted in the
Section of Seed Science and Technology, Indian Institute of Horticultural Research,
Hessaraghatta, and Bangalore-560089 March 2014 to September 2014. The details of the
materials used and methods adopted for the conduct of various experiments on seed
invigorationaredescribed hereunder:
Source of seeds: Freshly harvested and graded cucumber seeds of cv. Green long was
obtained from the seed production unit of IIHR, Hessaraghatta, Bangalore
The investigation was carried to know the biochemical basis of seed enhancement due to seed
invigoration.The different observations recorded are:
1.Electrical conductivity (dSm-1)
2.Total soluble seed protein (µg/ml)
3.Total soluble sugars ((µg/ml)4.
Total dehydrogenase activity (OD)
5. Amylase activity (µg starch hydrolyzed /ml/minute)
6.Catalase activity ( OD / minute)
7.Protein profile- SDS- PAGE
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8.Isozyme profile- NATIVE PAGE
3. Results and Discussion
3.1 Electrical Conductivity (Membrane Injury)
Dearman et al. (1986) reported that osmopriming of onion seeds with PEG-6000 at –1.5 MPa for 10
days at 150C significantly decreased the conductivity of seed leachate (98 to 28 µScm-1) over
unprimed seeds. The effect of priming on electrical conductivity in fresh and aged seeds of
cucumber is presented in figure 1. In case of fresh and aged seeds priming reduced the EC values
significantly. Compared to unprimed and hydro primed, priming with KH2PO4 and K2HPO4
reduced the electrical conductivity significantly. Similarly in aged seeds also all priming treatment
reduced the electrical conductivity. priming treatment applied to cucumber seeds after accelerated
ageing, improved germination by inducing protein hydrolysis, increased dehydrogenase enzymatic
activities and by decreasing electrolyte leakage (Habdas et al., 2000). When compared to hydro
priming, priming with potassium salts reduced more significantly the electrical conductivity. Pallavi
(2004) observed that osmopriming of cauliflower seeds with PEG-6000 at –1.0MPa for 72 hours
significantly enhanced the germination (84 to 94 %), mean seedling dry weight (2.34 to 3.13 mg),
SVI based on mean seedling dry weight (1009 to 1116) and decreased electrical conductivity of seed
leachate (196 to 159 dSm-1). Electrical conductivity of seed leachate is considered as a simple and
most reliable index of seed quality. In the present studies aged seeds showed 56.3 % higher electrical
conductivity compared to unaged indicating there is damage to the membrane leading to loss of
more solutes. Both high vigor and less viable seeds responded positively to the priming treatments
but more so in priming with potassium salt. Due to priming with K2HPO4 there was 33 and 44%
reduction in electrical conductivity when compared to control in high viable and less viable seeds,
respectively.
3.2 Total Soluble Sugars and Total Soluble Protein
The effect of priming on total soluble sugars and total soluble protein in high viable and less
viable seeds in cucumber is presented in fig 2 and 3. Both total soluble sugars and total
soluble protein in seed leachate were found high in case of less viable seeds compared to high
viable seeds indicating the loss of membrane integrity disturb the selective permeability of
the solutes enhance the leakage of more amount of the solutes in the leakage. Bourgne et al.
(2000) noticed significant increase in soluble protein content of individual primed seeds as
compared to the untreated sugar beet seeds (42.1to 232.8 mg/seed)
Jungmoon and suksoon (2004) reported that osmopriming of sweet corn seeds with –1.2 MPa
of PEG 8000 solution at 150C for 2 days resulted in increased -amylase activity, total soluble
sugars and decreased electrolyte leakage in such seeds. There was 58 and 12 % higher total
soluble sugars and total soluble protein, respectively, in less viable seeds compared to high
viable seeds. Although, the priming reduced the total soluble sugars values but these values
are almost on par with each other. In case of high viable seeds, however significant decrease
in total soluble sugars was noticed due to hydro priming as well priming with KH2PO4 and
K2HPO4. This reduction to the extent of 4.2, 8.95 and 10.6 respectively. Similar trend was
noticed with respect to total soluble protein also.
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3.3 Amylase Activity
Zuo et al. (1988a) reported that amylase activity during germination of primed peas and
tomato seeds. The effect of seed priming on amylase activity in fresh and aged seeds of
cucumber is presented in figure 1. Metabolic enzyme plays a key role in degrading seed
storage reserve in to simpler and transportable form to supply and nourishment to the
growing embryonic axis. Amylase is as such enzyme which breakdown starch in to the most
common transportable form of sugar. In the present investigation, the less viable seeds
showed 2.11 folds lower amylase activity compared to high viable seeds. Both high and less
viable seeds responded to the priming treatments. Upon priming with water, KH2PO4 and
K2HPO4, the percent increase in amylase activity in low vigor seeds was 1.5, 1.9 and 2.1
respectively. The high viability and vigor due to priming (table 1 and 2) might be activation
of this enzyme during priming process. Suksoon and Jaehyeum (2000) noticed that
osmopriming of rice seeds with PEG solution at –0.6 MPa for 4 days at 150C increased the -
amylase activity and they also reported that amylase activity was positively correlated with
the total soluble sugar content in such primed seeds.
3.4 Catalase Activity
Jeng and Sung (1994) found that free radicle scavenging enzymes such as superoxide
dismutase, catalase, and peroxidase and glyoxysome enzymes such asisocitrate lyase and
malate synthasewere increased by priming. The effect of seed priming on catalase activity in
fresh and aged seeds of cucumber is presented in figure 2. Catalase activity is generally
studied to understand an antioxidant scavenging capacity in the living tissue. These enzymes
tend to fluctuate during production of super oxide radical or single oxide molecules. In the
present investigation, the low vigor seeds showed higher catalase activity (38%) more
indicating the requirement of anti-oxidation to counteract the oxidation that occurred during
loss of vigor upon ageing. Although there was slight increase in less vigor seeds due to
priming process but no clear trend was noticed. Afzal et al. (2009) reported that germination
and seedling vigour can be enhanced by priming with Spermidine and Spermine in tomato
cultivars through maintaining higher level of antioxidant enzymes like superoxide dismutase
and catalase for eliminating excessive total peroxide.
3.5 Dehydrogenase Activity
Saha et al. (1990) reported that moisture equilibration soaking– drying treatment of soybean
seeds increased the total dehydrogenase activity and amylase activity (0.48 to 0.53 and 33 to
35 µg starch hydrolyzed hour-1seed-1). The effect of priming on dehydrogenase activity in
fresh and aged seeds of cucumber is presented in figure 3. All the living seeds those respire
produce enzymes called dehydrogenase hence, dehydrogenase activity is considered as the
positive biomarker for testing the seed viability status. In the present investigation less viable
seeds show reduced dehydrogenase activity due to death of viable tissue. This was to the
extent of 28.8% lower when compared to high viable seeds. In both high viable and less
viable seeds the dehydrogenase activity was increased significantly due to priming. In case of
high viable seeds the increase in dehydrogenase activity was to the extent of 48, 62 and 50 %
respectively. Due to hydro priming, KH2PO4 and K2HPO4, this was to the tune of 111, 121
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and 115% respectively. The increase dehydrogenase activity due to priming might be due to
enzyme activity initiation during priming and also high cell cycle activity during priming
process
3.6 Protein Profiles –Cucumber
Gurusinghe et al. (2002) used the seeds of tomato which were subjected to osmopriming with
PEG and subsequently dried at 37 or 400C for 2 to 4 hours to study the banding pattern of
total soluble seed proteins using SDS–PAGE. The effect of seed priming on protein profiles
in fresh and aged seeds of cucumber is presented in figure 4. Variation in protein profiles
were noticed between high vigor and less vigor seeds. High vigor seeds contain a total
number of 6 bands against only 4 bands in low vigor seeds. In both high and low vigor seeds
a common band was observed at Rm value 0.223 and 0.769. In less vigor seeds two
additional protein bands were observed at Rm value 0.861 and 0.915. These additional low
molecular weight bands might be due to breakdown of existing high molecular weight
proteins or due to synthesis heat shock related proteins. Hameed et al. (2010) reported the
expression of priming induced proteins (PIP) in wheat leaves, 36.7 kDa peptide, which was
expressed by all priming treatments such as hydro priming, freezing (-200C), 100 mM
CaCl2.2H20, 0.28 mM Ascorbate. In case of fresh and aged seeds, priming resulted in
synthesis of lost protein and also synthesis of some new proteins. In low vigor seeds, hydro
priming and showed five additional bands compared to unprimed. Whereas priming with
potassium salts showed bands up to maximum of 12 numbers besides variation in the number,
marked variation in the intensity of bands was also noticed due to priming in less vigor seeds
a common band at Rm value 0.915 was noticed. This can be used as a marker for seed
priming in cucumber.
3.7 Esterase Profile-Cucumber
The effect of seed priming on protein profiles in fresh and aged seeds of cucumber is
presented in figure 14. Variation in protein profiles were noticed between high vigor and less
vigor seeds. High vigor seeds contain a total number of 7 bands against only 2 bands in low
vigor seeds. In both high and low vigor seeds a common band was observed at Rm value
0.245 and 0.505. In higher vigor seeds additional protein bands were observed at Rm
value0.650 and 0.820. These additional bands might be due to breakdown of existing high
molecular weight proteins or due to synthesis heat shock related proteins. In case of fresh and
aged seeds, priming resulted in synthesis of lost protein and also synthesis of some new
proteins. In low vigor seeds, K2HPO4 priming showed 1 additional bands compared to
unprimed. Whereas priming with potassium salts showed bands up to maximum of 11
numbers besides variation in the number, marked variation in the intensity of bands was also
noticed due to priming in less vigor seeds a common band at Rm value 0.820 was noticed.
This can be used as marker for seed priming in cucumber.
4. Conclusion
Seed priming enhanced viability and vigor of low vigor seed and facilitated germination even
under adverse conditions. Hence the technique of seed priming can be adopted as a simple
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and cost effective technology for better crop stand establishment and higher productivity. The
present studies have great practical utility particularly in the present scenario of global
climate change and also in the context of safe seed storage and conservation (both formal and
informal) in gene banks.
Acknowledgements
The author would like to thank with his deep sense of gratitude and heartfelt respects to his
mentor Dr. K. Bhanuprakash, Principal Scientist, Seed Science and Technology section,
Indian Institute of Horticultural Research, Hessaraghatta, Bangalore for his valuable
suggestions, and guidances and also all the facilities during the whole period of research
Special thanks goes to NAM – Centre, Department of Science and Technology, Government
of India, New Delhi for providing fellowship to have great exposure for my better career. A
lot of thanks to the Head, section of Seed Science and Technology Dr. L. B. Naik and
Principal Scientist Dr. H.S. Yogesha, for their valuable guidance and support throughout the
research.
Table. 1 Effect of seed priming on electrical conductivity, soluble sugar and soluble protein
in fresh and aged seeds of cucumber.
EC (ds.m-1)
Total Sugar Total Soluble
(µg/ml)
Protein (µg/ml)
Treatment Fresh Aged Fresh Aged Fresh Aged
Control 225 400 197 313 152 181
Hydro Priming 200 315 190 300 150 170
KH2PO4 153 220 190 285 148 165
K2HPO4 150 223 190 280 151 165
Treatments: T1-Control, T2-Hydro priming,[email protected], T4- [email protected]
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Fig.1 &2 Effect of seed priming on amylase and catalase activity in fresh and aged seeds of
cucumber
Treatments: T1-Control, T2-Hydro priming,[email protected], T4- [email protected]
Fig. 3 Effect of seed priming on dehydrogenase activity in fresh and aged seeds of cucumber
Treatments: T1-Control, T2-Hydro priming,[email protected], T4 [email protected]
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BAND NO. RM VALUE T1 T2 T3 T4 T5 T6 T7 T8
1 0.223 + - + + + + +
2 0.323 + +++ + + - + +
3 0.407 + + + ++ - +++ + +
4 0.423 + ++ ++ + - + + +
5 0.600 - + + + - + + +
6 0.615 ++ + + + - + +++ +++
7 0.730 - - + +++ - + + -
8 0.769 ++ +++ +++ + ++ +++ +++ +
9 0.861 - + + + + + + +
10 0.915 - + + + + +++ +++ +++
11 0.938 - +++ +++ + - +++ +++ +
Fig. 4 Protein profile as influenced by priming in fresh and aged seeds of cucumber
Vigor Levels: V1- Fresh seeds - high vigor (>90%); V2- low vigor (<50%);
Treatments: Fresh seeds: T1-Control; T2-Water; [email protected], T4- [email protected]
Aged seeds:T5-Control; T6-Water; [email protected], T8- [email protected]
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BAND NO. RM VALUE T1 T2 T3 T4 T5 T6 T7 T8
1 0.166 - - +++ ++ - - - -
2 0.245 +++ +++ +++ ++ + + +++ +
3 0.249 + + + + - ++
4 0.395 + +++ +++ ++ - ++ +++ ++
5 0.456 +++ +++ +++ +++ - - + +++
6 0.505 + +++ + ++ + + +++ +
7 0.509 - + ++ - -
8 0.650 + ++ + +++ - + ++ ++
9 0.820 +++ - + - - - +++ +++
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10 0.910 - - - - - ++ +++ ++
11 0.950 - - - - - +++ - -
Fig. 5 Esterase Isozyme profile as influenced by priming in fresh and aged seeds of cucumber
Vigor Levels: V1- Fresh seeds - high vigor (>90%); V2- low vigor (<50%);
Treatments: Fresh seeds: T1-Control; T2-Water; [email protected],
Aged seeds:T5-Control; T6-Water; [email protected], T8- [email protected]
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